All lanes : Anti-alpha 2 Macroglobulin antibody (ab58703) at 1/1000 dilution

Lanes 1-3 : lysates prepared from rat spinal cord tissues (control group)
Lanes 4-6 : lysates prepared from rat spinal cord tissues (encephalomyelitis group)

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : goat anti-rabbit IgG coupled to horseradish peroxidase

Predicted band size: 163 kDa
Observed band size: 163 kDa
Additional bands at: 60 kDa (possible cleavage fragment), 80 kDa (possible cleavage fragment)



Three independent control and encephalomyelitis samples were used for Western blotting analysis. Samples were first resolved on a 12.5% SDS-PAGE gel and then transferred to a nitrocellulose membrane. The membrane was rinsed with PBS and the non specific binding sites were blocked in a solution of 5% non-fat milk in PBST (0.05% Tween 20 in PBS) for 1 hour at room temperature, followed by three washes in PBST for 10 minutes each. The membrane was first incubated with ab58703 at a 1/1,000 dilution overnight and then washed in PBST buffer as described above. The immunocomplexes were visualized by Western Lightning® Western Blot Chemiluminescence Reagent Plus, using a goat anti-rabbit IgG coupled to horseradish peroxidase as the secondary antibody.

Ab58703 staining alpha 2 Macroglobulin in human liver.
Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffers EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.